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PubMed2026

Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement.

The development of CRISPR multiplex genome-editing (MGE) tools is rapidly transforming plant functional genomics and accelerating crop improvements. By simultaneously targeting two or more DNA loci, it allows scientists to precisely edit multiple genes at the single-nucleotide level, within the target genome. Simultaneous manipulation of multiple targets has revolutionized the functional elucidation studies, particularly the dissection of complex genetic pathways. Due to its superior precision and feasibility, CRISPR-MGE is widely accepted and has largely replaced alternative editing tools such as TALENs and ZFNs. Several CRISPR-MGE strategies, including the use of individual expression cassettes, tRNA-processing enzymes, Csy4 or ribozymes, have been successfully deployed in plants. Recent advancements, such as Cpf1, transgene-free methods, or ultra-multiplexing approaches, have further refined the technology into a powerful, efficient, and robust toolkit. MGE enables complex genome editing, including multiple-gene knockouts, base alterations, transcriptional regulation, metabolic engineering, or their combinations. Consequently, it is ideal for elucidating the function of transcription factors that are key molecular players in regulating diverse plant responses, especially in stress pathways. Several stress-responsive TFs have been functionally characterized via CRISPR-MGE, and more advanced tools are being employed. This review evaluates multiplexing tools, their diverse applications, and the current progress toward developing advanced MGE tools. Ultimately, we provide evidence to encourage the use of advanced MGE tools for functional characterization studies of stress-responsive TFs, thereby highlighting their potential to accelerate crop improvement.

Gene Editing↗
PubMed2026

An efficient woody plant protoplast platform enables transgene-free multiplex genome editing and rapid trait validation in pear.

Multiplex editing is crucial for analysing complex multiple-gene traits in woody plants, yet its application remains limited because of low transformation efficiency and lengthy regeneration cycles. To overcome these barriers, in this study we establish an efficient protoplast isolation protocol for pear (Pyrus) that employs 1.0% cellulase R10 and 0.4% macerozyme R10 with an 8.5 h digestion. Its broad applicability using different digestion times across seven other economically important woody plants is demonstrated. Coupling a 40% PEG-4000-mediated transfection regimen with DNA-free CRISPR/Cas9 ribonucleoprotein (RNP) delivery enables multiplex genome editing in isolated protoplasts. Using this platform, we simultaneously disrupted the key components of the chloroplast division apparatus ARC3, PARC6, and FtsZ2-1a in Pyrus bretschneideri and found that it consistently reproduced macro-chloroplast abnormalities, confirming effective multigene perturbation within a single cellular context. Notably, failure of chloroplast division activated chloroplast-to-nucleus retrograde signaling, as evidenced by the induction of the nuclear stress-response genes RBOHD and ZAT12, a concomitant surge in reactive oxygen species, and progression to severe cellular deformation. Thus, our study establishes a rapid, cross-genus protoplast-RNP workflow that enables DNA-free multiplex editing and accelerates genotype-to-phenotype analyses in woody perennials. The approach provides a practical foundation for functional genomics and supports advances in non-transgenic precision breeding of tree crops.

Protoplasts↗
PubMed2025

Development of a multiplex PCR for detection of pathogenic Mycobacterium orygis in cattle tissues harboring tuberculous-like lesions.

Mycobacterium orygis, a recently defined member species of Mycobacterium tubercuolsis complex (MTBC), is emerging as a major threat to zoonotic tuberculosis control, especially in the Asian Subcontinent. The dearth of low-cost diagnostic assay to differentiate M. orygis from other members of the MTBC leads to unavailability of information about the actual burden of this species in human and animal population. In this study, we developed a multiplex PCR for distinguishing M. orygis from other MTBC based on two M. orygis-specific nonsynonymous point mutations in mbtG and fadD23 genes identified by comparative genome analysis. The specificity of the assay shows that a 434 bp IS1081 fragment was amplified from common MTBC species including M. orygis while 240 bp and 181 bp mbtG and fadD23 gene fragments were amplified only from M. orygis. No amplification was observed for nontuberculous Mycobacterium (NTM) and non-Mycobacterial pathogens. The multiplex PCR assay showed a detection limit of 32 pg of M. orygis DNA. Furthermore, a total of 85 tuberculous-like lesions in the different tissues of slaughtered cattle were tested for identification of the M. orygis, and the results showed IS1081, mbtG and fadD23 amplicons in three tissue DNA extracts confirming they contain M. orygis DNA. Also, a single IS1081 amplicon was amplified from one tissue sample signifying presence of DNA of any MTBC species other than M. orygis. An established TaqMan real time PCR assay targeting region of differences (RD) in M. orygis genome was carried out to validate the result of the assay. This showed 100 % accuracy of the in-house developed multiplex PCR.

Mycobacterium orygis↗
PubMed2001

Rapid detection by fluorescent multiplex PCR of exon deletions and duplications in the C1 inhibitor gene of hereditary angioedema patients.

Hereditary angioedema (HAE) is due to a variety of defects in the C1 inhibitor gene (C1NH gene), including approximately 20% of partial deletions/duplications whose boundaries are usually within Alu repeats. To ensure complete molecular characterization of C1 inhibitor deficiencies a fluorescent multiplex assay was constructed to amplify simultaneously five exons of C1NH and an exon of the BRCA1 gene. PCR protocols were optimized for these amplicons (size range between 300 and 700 bp). Forward and reverse chimeric primers that carry strand-specific 5' tags of 16 nucleotides were used to ensure similar levels of PCR products for each amplicon in the multiplex. Data were analyzed by superposing fluorescent profiles of test and control DNA and by visually comparing the normalized peak levels of corresponding amplicons, rather than by calculating the ratios of peak areas. Tests on a collection of known defects, including five different Alu-mediated deletions and a partial duplication have validated this approach. In a study of 19 sporadic cases of HAE, of which four had failed to reveal mutations upon screening all exons by fluorescent chemical cleavage, three de novo deletions were diagnosed by using this multiplex PCR approach: a deletion of exon 4, a deletion of exons 5 and 6, and an apparently complete gene deletion. Besides being suitable for the initial DNA screening of the C1NH gene in HAE patients prior to screening for point mutations, this method can be easily adapted to complex genes for the screening of rearrangements.

Angioedema↗
PubMed2001

Multiplexed enzyme assays in capillary electrophoretic single-use microfluidic devices.

We describe a method of performing multiple enzyme assays in a single reaction vessel. The resolving power of capillary electrophoresis enables several enzyme assays to be analyzed at high speed in microfluidic arrays. Multiplexed measurement can increase throughput significantly without requiring highly dense microfluidic arrays. Enzyme assays in a multiplexed format for selected kinases in this work show essentially identical performance to assays performed individually. This establishes an approach for screening one compound against multiple enzyme targets simultaneously. Another potential application for performing multiplexed enzyme assay is to study protein-protein (especially enzyme-enzyme) interaction by monitoring the enzymatic activity changes.

Amino Acid Sequence↗
PubMed2002

Stratification of pedigrees multiplex for systemic lupus erythematosus and for self-reported rheumatoid arthritis detects a systemic lupus erythematosus susceptibility gene (SLER1) at 5p15.3.

OBJECTIVE: Arthritis is a common manifestation in systemic lupus erythematosus (SLE), appearing in approximately 85% of patients. Often, the polyarthritis at presentation of SLE cannot be distinguished from rheumatoid arthritis (RA) by physical examination or history. Indeed, physicians initially tell many SLE patients that they have RA (one source of "self-reported RA"), only to have SLE established later. In addition, RA aggregates in families with an SLE proband. We predicted that pedigrees multiplex for both SLE and for self-reported RA would better isolate particular genetic effects. If this proved to be true, we would then use the increased genetic homogeneity to more easily reveal genetic linkage. METHODS: From a collection of 160 pedigrees multiplex for SLE, we selected 36 pedigrees that also contained >or=2 members with self-reported RA (19 pedigrees were African American, 14 were European American, and 3 were of other ethnic origin). Data from a genome scan of 307 microsatellite markers were evaluated for SLE linkage by contemporary genetic epidemiologic techniques. RESULTS: The most significant evidence of linkage to SLE was obtained at 5p15.3 in the European American pedigrees by both parametric (logarithm of odds [LOD] score 6.2, P = 9.3 x 10(-8)) and nonparametric (LOD score 6.9, P = 1.7 x 10(-8)) methods. The best-fitting model for this putative SLE gene in this region was a recessive gene with a population frequency of 5% and with 50% penetrance in females and 15% penetrance in males at virtually 100% homogeneity. CONCLUSION: For a genetically complex disease phenotype, an unusually powerful linkage has been found with SLE at 5p15.3 in European American pedigrees multiplex for SLE and for self-reported RA. This result predicts the presence of a gene at the top of chromosome 5 in this subset of patients that is important for the pathogenesis of SLE.

Arthritis, Rheumatoid↗
PubMed2005

Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution.

A new and convenient method for the in vitro recombination of single point mutations is presented. This method efficiently reduces the introduction of novel point mutations, which usually occur during recombination processes. A multiplex polymerase chain reaction (multiplex-PCR) generates gene fragments that contain preformed point mutations. These fragments are subsequently assembled into full-length genes by a recombination-PCR step. The process of multiplex-PCR-based recombination (MUPREC) does not require DNase I digestion for gene-fragmentation and is therefore easy to perform, even with small amounts of target DNA. The protocol yields high frequencies of recombination without creating a wild-type background. Furthermore, the low error rate results in high-quality variant libraries of true recombinants, thereby minimizing the screening efforts and saving time and money. The MUPREC method was used in the directed evolution of a Bacillus subtilis lipase that can catalyse the enantioselective hydrolysis of a model meso-compound. Thereby, the method was proved to be useful in producing a reliable second-generation library of true recombinants from which better performing variants were identified by using a high-throughput electrospray ionization mass spectrometry (ESI-MS) screening system.

Amino Acid Sequence↗
PubMed2004

Multiplexed, particle-based detection of DNA using flow cytometry with 3DNA dendrimers for signal amplification.

BACKGROUND: Complex mixtures of DNA may be found in environmental and medical samples. There is a need for techniques that can measure low concentrations of target DNAs. For a multiplexed, flow cytometric assay, we show that the signal-to-noise ratio for fluorescence detection may be increased with the use of 3DNA dendrimers. A single fluorescent DNA molecule per bead could be detected with conventional flow cytometry instrumentation. METHODS: The analyte consisted of single-stranded (ss) DNA amplicons that were hybridized to capture probes on the surface of fluorescent polystyrene microspheres (beads) and initially labeled with streptavidin-R-phycoerythrin (single-step labeling). These beads have a low reporter fluorescence background and high efficiency of DNA hybridization. The DNA/SA-RPE complex was then labeled with 3DNA dendrimers and SA-RPE. The bead complexes were detected with a Luminex 100 flow cytometer. Bead standards were developed to convert the intensity to the number of SA-RPE labels per bead and the number of dendrimers per bead. RESULTS: The dendrimer assay resulted in 10-fold fluorescence amplification compared with single-step SA-RPE labeling. Based on concentration curves of pure target ss-amplicons, the signal-to-noise ratio of the dendrimer assay was greater by a factor of 8.5 over single-step SA-RPE labeling. The dendrimer assay was tested on 16S ribosomal DNA amplified from filter retentates of contaminated groundwater. Multiplexed detection of a single dendrimer-labeled DNA molecule per bead was demonstrated. CONCLUSIONS: Multiplexed detection of DNA hybridization on a single molecule level per bead was achieved with conventional flow cytometry instrumentation. This assay is useful for detecting target DNAs at low concentrations.

Calibration↗
PubMed2006

Multiplexed and microparticle-based analyses: quantitative tools for the large-scale analysis of biological systems.

While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach. The past few years have seen an explosion in the application of flow cytometry technology for molecular analysis and measurements using microparticles as solid supports. While microsphere-based molecular analyses using flow cytometry date back three decades, the need for highly parallel quantitative molecular measurements that has arisen from various genomic and proteomic advances has driven the development in particle encoding technology to enable highly multiplexed assays. Multiplexed particle-based immunoassays are now common place, and new assays to study genes, protein function, and molecular assembly. Numerous efforts are underway to extend the multiplexing capabilities of microparticle-based assays through new approaches to particle encoding and analyte reporting. The impact of these developments will be seen in the basic research and clinical laboratories, as well as in drug development.

Coloring Agents↗
PubMed1998

Analysis of multiplexed short tandem repeat (STR) systems using capillary array electrophoresis.

The profiling of polymorphic short tandem repeat (STR) markers is being applied to human identification, parentage testing and genetic mapping. Reliable genotyping of these markers is facilitated by polymerase chain reaction (PCR) amplification and high-resolution electrophoretic separation. Capillary array electrophoresis (CAE) offers very rapid, high-resolution separation of the amplified DNA and potential for automated sample processing not realized employing conventional slab-gel electrophoresis. The use of CAE to type DNA samples amplified at 11 genetic loci in multiplex profiles is presented. Two sets totaling 208 samples were amplified in a multiplex fashion using AmpFlSTR-Blue or AmpFlSTR-Green I and analyzed in a blind study using CAE. With the exception of one sample, the CAE genotyping results were in complete agreement with results obtained using a single-capillary system or two slab-gel electrophoresis systems. The sample, genotype TH01 7/10, migrated similar to TH01 6.3/9.3 allele sizes, which suggested a potential band migration shift. The recommended approach to such an observation is to analyze the sample again. The sample was rerun and correct genotype verified. Allelic ladder samples were analyzed multiple times by CAE to determine sizing accuracy and precision. The sizing of over 240 allelic ladder samples yielded an average within-run precision of +/- 0.13 bp and between-run precision of +/- 0.21 bp for fragments up to 350 bp. The CAE protocols permit processing of up to 96 multiplex STR samples in under 70 min.

Alleles↗
PubMed2005

A qualitative look at multiplex gene expression of single cells using capillary electrophoresis.

We demonstrate the first use of capillary electrophoresis with laser-induced fluorescence (CE-LIF) for the qualitative analysis of single-cell multiplex products of the reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of both estrogen receptor alpha (ERalpha) and beta-actin in individual MCF-7 cells was monitored using a one-pot reaction. Reverse transcription and a single round of touch-down PCR, performed in a multiplex format, were used to generate fragment sizes of 318 bp and 838 bp, for ERalpha and beta-actin, respectively. A replaceable hydroxypropylmethylcellulose sieving matrix was used to effect a size-based separation of ethidium bromide-bound DNA. As titration of RT-PCR reaction components did not appreciably influence multiplex product generation, the use of additives, including bovine serum albumin (BSA) and herring sperm DNA, was explored. The addition of BSA to the RT-PCR mixture only resulted in efficient amplification of beta-actin, whereas the DNA carrier allowed co-amplification of both ERalpha and beta-actin. Furthermore, the sensitivity of our CE-LIF method eliminated the need for a second round of nested PCR, typically required when RT-PCR products are analyzed using gel electrophoresis.

Actins↗
PubMed2005

Multiplex amplified product-length polymorphism analysis of 36 mitochondrial single-nucleotide polymorphisms for haplogrouping of East Asian populations.

We present a reliable, rapid, and economical multiplex amplified product-length polymorphism (APLP) method for analyzing the haplogroup-diagnostic mitochondrial single-nucleotide polymorphisms (mtSNPs) in East Asian populations. By examining only 36 haplogroup-specific mtSNPs in the coding region by using four 9-multiplex polymerase chain reaction (PCR) and subsequent electrophoresis, we could safely assign 1815 individuals from 8 populations of Japanese, Korean, Chinese, and Germans to 45 relevant haplogroups. This multiplex APLP analysis of coding-region mtSNPs for haplogrouping is especially useful not only for molecular phylogenetic studies but also for large-scale association studies due to its rapid and economical nature. This is the first panel of mtSNPs in the coding region to be used for haplogrouping of East Asian populations.

Asian People↗
PubMed2006

Dimethyl multiplexed labeling combined with microcolumn separation and MS analysis for time course study in proteomics.

Stable-isotope labeling coupled with liquid-phase separation and MS analysis is a powerful technique for comparative proteomics. We developed a dimethyl labeling strategy (Anal. Chem. 2003, 75, 6843-6852 and J. Proteome Res. 2005, 4, 101-108) to label peptide N-terminus and epsilon-amino groups of Lys with water-soluble formaldehyde via reductive methylation, and an isotopic pair of formaldehyde is used for binary labeling on two sets of samples. In this study, this approach is extended to a four sample labeling by combining the binary isotopic reagents of formaldehyde (d0, d2) and the binary isotopic reducing reagents, sodium cyanoborohydride (d0, d3). To ensure sufficient mass difference, this multiplexed labeling is coupled with endoproteinase Lys-C instead of trypsin for digestion, resulting in at least two labeling sites with a mass difference of 4 Da for each pair of peptide digest. Moreover, multiplex dimethyl labeling was proved to have no significant isotopic effect during RP LC elution. This method was further applied for monitoring Lys-C digestion using hemoglobin as a model. Dimethyl labeled digests derived from seven time points (1-30 h) were grouped into two sets of sample mixtures, separated by nano-LC to reduce the complexity, and then analyzed by ESI-MS/MS. The temporal study reveals that Lys-C digestion was completed in 10-15 h for all detected peptides. The multiplex dimethyl method has not only provided a simultaneous detection mean for four sample sets but has also conserved all the advantages associated with the original binary method.

Amino Acid Sequence↗
PubMed2002

Highly multiplexed genotyping of coronary artery disease-associated SNPs using MALDI-TOF mass spectrometry.

Highly multiplexed genotyping methods are needed to support a comprehensive analysis of single nucleotide polymorphisms (SNPs) in coronary artery disease (CAD)-related genes. In this study we evaluated chip-based MALDI-TOF mass spectrometry for multiplexed genotyping of SNPs associated with CAD. Our analysis included 14 healthy Japanese individuals and 19 Japanese patients with myocardial infarction whose first attack occurred before age 50. We selected 29 candidate genes involved in 1) the renin-angiotensin system, 2) lipid metabolism, 3) cytokines and adhesion molecules, 4) growth factors, and 5) the coagulation-fibrinolysis system. Genotyping of candidate SNPs was performed by MALDI-TOF MS using a MassARRAY system, and 4-plex analysis was achieved at a maximum. All 39 SNPs determined by the fluorescent dye-terminator cycle sequencing method from four randomly selected patients were found to be in complete agreement with the results obtained from MassARRAY system. Significant differences were observed in the -1965delG of PAI1 (SERPINE1) with respect to allelic frequency, the G>A in the promoter region SNP in SM22 (TAGLN) for dominant genotype, and in two other SNPs (C>T in intron 1 of HGF, and -1965delG of PAI1) for recessive genotype. Three SNPs (803T>C of AGT, 677CT of MTHFR, 190T>C of ADRB3) showed weak differences in allelic frequency. MALDI-TOF-MS provided high performance with a multiplex assay design for analysis of CAD-related SNPs by increasing the throughput while maintaining a high level of accuracy.

Cell Adhesion Molecules↗
PubMed2002

Rapid detection of novel BRCA1 rearrangements in high-risk breast-ovarian cancer families using multiplex PCR of short fluorescent fragments.

Recent studies have revealed a significant proportion of BRCA1 exon deletions or duplications in breast-ovarian cancer families with high probability of BRCA1- or BRCA2-linked predisposition, in which mutations of these genes have not been found. The difficulty of detecting such heterozygous rearrangements has stimulated the development of several new screening methods. Quantitative fluorescent multiplex PCR is based on simultaneous amplification of multiple target sequences under conditions that allow rapid and reliable quantitative comparison of the fluorescence of each amplicon in test samples and in controls. The modified method described here, named quantitative multiplex PCR of short fluorescent fragments (QMPSF), is particularly well suited for large genes. All BRCA1 coding exons were analyzed using four multiplexes in 52 families without point mutations in the exons or splice-sites of BRCA1 and BRCA2, and selected because of high probability of a BRCA1- or BRCA2-linked genetic predisposition. Five distinct BRCA1 rearrangements were detected: a deletion of exons 8-13, a duplication of exons 3-8, a duplication of exons 18-20, a deletion of exons 15-16, and a deletion of exons 1-22-which is the largest deletion found so far within the BRCA1 gene. The method described here lends itself to rapid, sensitive, and cost-effective search of BRCA1 rearrangements and may be included into the routine molecular analysis of breast-ovarian cancer predispositions. Hum Mutat 20:218-226, 2002.

BRCA1 Protein↗
PubMed1995

Point mutation screening for 16 exons of the dystrophin gene by multiplex single-strand conformation polymorphism analysis.

We have developed a rapid and nonradioactive method to screen for point mutations using the Pharmacia PhastSystem. In an SSCP analysis, we applied the two multiplex exon PCR kits, commonly used for the detection of deletions in Duchenne and Becker muscular dystrophy patients. The different exon bands in the multiplex SSCP pattern could be identified by running well-characterised deletion patients in this system. Two common polymorphisms were easily identifiable and are helpful in the haplotype analysis in families. Screening of 70 patients in which no gross rearrangement was detectable with the multiplex PCR and Southern blot, resulted in the identification of 6 patients with a band shift after SSCP analysis. Of these 6 band shifts, 5 were the result of a frame shift or termination mutation. The other band shift was found to be a rare polymorphism unlikely to be the cause of the patient's phenotype. Application of this technique enabled us to improve diagnosis in the families involved and will allow us to extend the search for point mutations in the remaining exons of the dystrophin gene.

Blotting, Southern↗
PubMed2005

Detection of cryptic subtelomeric chromosome abnormalities and identification of anonymous chromatin using a quantitative multiplex ligation-dependent probe amplification (MLPA) assay.

The need to detect clinically significant segmental aneuploidies beyond the range of light microscopy demands the development of new cost-efficient, sensitive, and robust analytical techniques. Multiplex ligation-dependent probe amplification (MLPA) has already been shown to be particularly effective and flexible for measuring copy numbers in a multiplex format. Previous attempts to develop a reliable MLPA to assay all chromosome subtelomeric regions have been confounded by unforeseen copy number variation in some genes that are very close to the telomeres in healthy individuals. We addressed this shortcoming by substituting all known polymorphic probes and using two complementary multiplex assays to minimize the likelihood of false results. We developed this new quantitative MLPA strategy for two important diagnostic applications. First, in a group of cases with high clinical suspicion of a chromosome abnormality but normal, high-resolution karyotypes, MLPA detected subtelomeric abnormalities in three patients. Two were de novo terminal deletions (del(4p) and del(1p)), and one was a derivative chromosome 1 from a maternal t(1p;17p). The range of these segmental aneuploidies was 1.8-6.6 Mb, and none were visible on retrospective microscopy. Second, in a group of six patients with apparently de novo single-chromosome abnormalities containing anonymous chromatin, MLPA identified two cases with simple intrachromosomal duplications: dup(6p) and dup(8q). Three cases showed derivative chromosomes from translocations involving the distal regions of 9q and 4q, 5p and 11q, and 6q and 3p. One case showed a nonreciprocal, interchromosomal translocation of the distal region of 10p-7p. All abnormalities in both groups were confirmed by fluorescence in situ hybridization (FISH) using bacterial artificial chromosomes (BACs). This quantitative MLPA technique for subtelomeric assays is compared with previously described alternative techniques.

Adolescent↗
PubMed2002

Evaluation of a single-step multiplex RT-PCR for influenza virus type and subtype detection in respiratory samples.

A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for the simultaneous detection of influenza virus type and subtypes was evaluated for its use in clinical specimens. One part of each specimen was tested using an established standard culture and/or monoclonal antibody-based immunofluorescence assay method, and the other part was tested by the multiplex RT-PCR method for the presence or absence of the influenza virus, and its type and subtype. Sixty-seven specimens were examined. The results revealed a strong agreement between the data obtained by the established method and those obtained by the multiplex RT-PCR.

Antigens, Viral↗